The molecular mechanism of muscle contraction is thought to involve some change in the myosin head while attached to actin (either a conformational change within the head itself or an alteration in the binding angle made with actin). A considerable amount is known about the structure of one attached state of myosin, the so-called strongly bound state, which occurs with bound ADP or no nucleotide. However, little is known about the structure(s) of other so-called weakly bound states because at low protein concentration the acto-myosin complex is almost completely dissociated by ATP. The initial aim of the work described in this proposal is to analyze the structure of these weakly bound actomyosin states using frozen hydrated electron microscopy. A second aim of this work is to study the structure of defined biochemical intermediates of the actomyosin ATP hydrolysis mechanisms using rapid- freeze quench electron microscopy. The predominant biochemical intermediate will be selected by varying either the time interval before freezing or experimental conditions (pH, nucleotide, temperature). In this manner we plan to determine how the structure of the actomyosin varies with time and biochemical state. Cryo-electron microscopy is especially well suited for this type of study as it allows extremely fast fixation and excellent preservation. Despite two decades of intensive investigations, knowledge of the structural changes accompanying force production remains the single most important problem in understanding normal muscle function. Such knowledge is also likely to be important for understanding and ameliorating pathological conditions in skeletal and cardiac muscle.